The goal of this research plan is to understand the mechanism by which synaptotagmin couples increases in intracellular Ca 2+ concentrations to the exocytotic fusion of secretory vesicles in living cells. Our hypothesis is that the Ca 2+ sensitivity of exocytosis for a given cell reflects the Ca 2+ affinities of the cells complement of synaptotagmin isoforms. To address this question, three Specific Aims are proposed: first, synaptotagmin-effector interactions that mediate Ca 2+ dependent secretion will be identified by elucidating the mechanism of inhibition of Ca 2+ dependent exocytosis in neuroendocrine PC12 cells by C2 domains from synaptotagmins I-XI. Preliminary data indicated that the C2 domains inhibit by disruption of endogenous synaptotagmin interactions with the lipid phosphatidyl-inositol-4,5-bisphosphate (PIP2) or the SNARE proteins SNAP25 and syntaxin. Second, the capacity of the synaptotagmin family to serve as Ca 2+ sensors over a wide range of Ca 2+ concentrations will be determined. Using biochemical and biophysical assay, the Ca 2+ dependency of synaptotagmin membrane and SNARE interactions will be determined for synaptotagmins I-XI. Finally, synaptotagmins identified with Ca 2+ affinities higher or lower than synaptotagmins I and IX, the predominate synaptotagmins in PC12 cells, will be expressed in the neuroendocrine cells to test whether they alter the Ca 2+ sensitivity of exocytosis. Thus, we will directly test our hypothesis that the Ca 2+ sensitivity of exocytosis for a given cell reflects the Ca 2+ affinities of the cell's complement of synaptotagmin isoforms. A better understanding of how the synaptotagmin family regulates neuronal exocytosis will contribute to the overall mechanism of synaptic transmission and how this process contributes to synaptic plasticity. An understanding of the regulation of neuronal exocytosis may ultimately provide targets for treatment of diseases in which synaptic transmission is impaired [unreadable] [unreadable]